The detection and imaging of molecules in biological systems is a vitally important tool in biomedical research. Conventional fluorescence microscopy is an extremely useful technique, but is limited by the loss of resolution caused by emission from sources outside of the focal plane. Confocal Laser Scanning Microscopy circumvents this problem by scanning the specimen with light from a laser-illuminated pinhole and imaging the emitted light through a detector pinhole. All out-of-focus fluorescence is rejected by the pinhole, resulting in submicron resolution in the X, Y, and Z axes. c In 1998, funds from a NCRR Shared Instrument Grant, a UAB Health Services Foundation award, and other resources from the UAB Office of the Provost, the Chairs of Departments in both the Medical and Academic Divisions of DAB, and Center Directors were used to establish, the High Resolution Imaging Facility. This shared resource is equipped with a Leica Confocal Imaging Spectrophotometer TCS SP unit configured for multiphoton excitation with a Coherent MIRA Ti:Sa ultrafast laser with output selectable between 690-990 nanometers This permits imaging of living cells and tissues with minimal phototoxicity, and of structures 200- 400 micrometers from the tissue surface. The SP allows user selection of specific portions of the emission spectrum to be delivered to the detector. The result is higher fluorescence yield, reduced crosstalk, and improved image quality. The Facility also includes an Olympus Fluoview CLSM with fiber launched Krypton ion and Argon ion lasers, conventional fluorescence and DIC microscopy equipment, and work stations equipped with digital deconvolution software, output and image storage devices. Through a cooperative agreement with the Birmingham Veteran's Administration Medical Center, RDCC members have access to a Leica TCS SP unit equipped with a UV laser and UV corrected optics. The Leica instruments are equipped with either three or four detector channels, a transmitted light channel, and Argon ion, Krypton ion and HeliunrNeon lasers, and are able to image up to three fluorophores simultaneously. In a cooperative agreement with the Cell Adhesion and Matrix research center, and the Vision Science Research Center, the HRIF now offers access to a state-of-the-art FRET system. Significant funds for new in vivo imaging instrumentation have been obtained through a successful NIH Blueprint application, and a Shared Instrument Grant application for high speed image acquisition capability is pending. The HRIF served 70 RDCC investigators during the current funding period and their use accounted for 49% of the total facility usage. During this same period, support from the RDCC P30 mechanism accounted for less than 20% of the total annual operating costs of the facility.